1. Field of the Invention
The invention relates generally to non-peptide molecules having affinity for the erythropoietin receptor at a modulating site that is other than the erythropoietin binding site and the use of such molecules.
2. Background Information
As the cellular pathways are discovered, there is increasing insight into the different molecules that are involved in the transduction of a signal resulting from the binding of a ligand to a cellular membrane receptor to the transcription and expression of genes in the host cell genome. The development of erythropoietin (“EPO”) has allowed for the substantial improvement in the treatment of anemia. Anemia may be a natural phenomenon resulting from a genetic defect or may be as a result of infection or treatment with radiation or chemotherapy. In each of these instances it is necessary for the viability of the host that the red blood cell count or hematocrit remain substantially normal. In many situations, the level of treatment is compromised by the adverse effect on the red blood cell count, so that the desired therapy cannot be administered. The availability of EPO has provided a means for restoring the red blood cell count in compromised patients.
EPO is a large glycosylated protein that is expensive and difficult to manufacture, purify and formulate. It is also degraded by proteases when in the blood, so that a substantial portion of the administered EPO is lost. Despite these problems, the dramatic effect achieved by EPO has resulted in annual sales exceeding a billion dollars.
In U.S. Pat. No. 6,333,031 and in Activation of erythropoietin Receptor Through a Novel Extracellular Binding Site, Naranda, et al Endocrinology 2002 143(6):2293–2302; Activation of Erythropoietin Receptor in the Absence of Hormone by a Peptide That Binds to a Domain Different from the Hormone Binding Site, Naranda, et al., Proc. Natl. Acad. Sci. 1999, 96(13):7569–74 are reported the existence of an extracellular binding site of the EPO receptor (“EPO-R”) referred to as the “modulation domain.” In the human EPO-R, the modulation domain corresponds to about amino acids 194–216 and has the amino acid sequence QRVEILEGRTECVLSNLRGRTRY (SEQ ID NO:1). Binding of a 23 amino acid peptide having the sequence SEQ ID NO:1 to the EPO-R modulating domain resulted in modulation of the activity of the EPO-R in the presence or absence of the ligand. The peptide, therefore, offers an auxiliary compound for use with EPO to enhance thje activity of EPO and reduce the requirement for EPO to achieve analogous activity. The peptide can be readily synthesized efficiently and economically. However, the peptide is small and will be rapidly degraded in the blood. Also, it suffers from the inconvenience of requiring injection.
There is, therefore, an interest in developing small non-peptide molecules that can serve in conjunction with EPO in the treatment of anemia, as well as in other functions associated with assays for EPO-R, studying EPO and EPO-R function, purification of EP0-R, and analogous uses.